The present invention relates to novel double transgenic non-human animals useful to model diseases involving amyloidopathies, in particular Alzheimer's disease. Such transgenic animals will have utility in developping specific and general therapies for the treatment of amyloidopathies and in screening methods to identify novel anti-amyloidogenic compounds. The present invention is further directed to a method for the generation of such transgenic animals, to the evaluation of the in vivo effects of beta-secretase activity on amyloid peptide generation, amyloidosis, neurodegeneration and Alzheimer's pathology through the use of such novel transgenic animals.
Alzheimer's disease (AD) is a neurodegenerative disease which is characterized by memory loss and decline of cognitive functions (McKhann G, Drachman D, Folstein M, Katzman R, Price D, Stadlan E M. Clinical diagnosis of Alzheimer's disease: report of the NINCDS-ADRDA Work Group under the auspices of Department of Health and Human Services Task Force on Alzheimer's Disease. Neurology. 1984.34:939–44). The prevalence of AD approximately doubles with every five years over the age of 65. Overall, approximately 5 to 10% of those over 65 are affected (Scorer C A. Preclinical and clinical challenges in the development of disease-modifying therapies for Alzheimer's disease. DDT. 2001.6: 1207–1219). The clinical diagnosis of AD remains an exclusion diagnosis. Only post-mortem microscopic examination of the brain offers a definitive diagnosis based on the presence of extracellular plaques containing fibrils of amyloid-beta (A-beta) peptide and intracellular tangles containing polymerized phosphorylated Tau protein (Glenner G G, Wong C W. Alzheimer's disease and Down's syndrome: sharing of a unique cerebrovascular amyloid fibril protein. Biochem Biophys Res Commun. 1984. 22:1131–5; Spillantini M G, Goedert M, Jakes R, Klug A. Different configurational states of beta-amyloid and their distributions relative to plaques and tangles in Alzheimer disease. Proc Natl Acad Sci U S A. 1990. 87:3947–51). In most of the cases, AD occurs late in onset and sporadically. The early onset familial AD represents a minority of the cases, but they have been extremely important for the progression in the understanding of the disease mechanisms. Mutations in three genes have been shown to cause early-onset familial AD (FAD): Amyloid Precursor Protein (APP) (Chartier-Harlin M C, Crawford F, Houlden H, Warren A, Hughes D, Fidani L, Goate A, Rossor M, Roques P, Hardy J, et al. Early-onset Alzheimer's disease caused by mutations at codon 717 of the beta-amyloid precursor protein gene. Nature. 1991. 353: 844–6; Goate A, Chartier-Harlin M C, Mullan M, Brown J, Crawford F, Fidani L, Giuffra L, Haynes A, Irving N, James L, et al. Segregation of a missense mutation in the amyloid precursor protein gene with familial Alzheimer's disease. Nature. 1991.349:704–6; Murrell J, Farlow M, Ghetti B, Benson M D. A mutation in the amyloid precursor protein associated with hereditary Alzheimer's disease. Science. 1991. 254: 7–9; Mullan M, Houlden H, Windelspecht M, Fidani L, Lombardi C, Diaz P, Rossor M, Crook R, Hardy J, Duff K, et al. A locus for familial early-onset Alzheimer's disease on the long arm of chromosome 14, proximal to the alpha 1-antichymotrypsin gene. Nat Genet. 1992. 2: 340–2), PresenilinI (Psenl) (Sherrington R, Rogaev E I, Liang Y, Rogaeva E A, Levesque G, Ikeda M, Chi H, Lin C, Li G, Holman K, et al. Cloning of a gene bearing missense mutations in early-onset familial Alzheimer's disease. Nature. 1995. 375:754–60), and Presenilin 2 (Psen2) (Levy-Lahad E, Wijsman E M, Nemens E, Anderson L, Goddard K A, Weber J L, Bird T D, Schellenberg G D. A familial Alzheimer's disease locus on chromosome 1. Science. 1995. 269:970–3). Known missense mutations affect codon 717 of APP (altering V717*1, V717+G and V717+F in the polypeptide), while codons 670/671 (altering K670+N and M671+L in the polypeptide, hereinafter referred to as the Swedish mutation) are altered in the APP gene of a Swedish AD pedigree (numbers according to APP770). All these mutations affect the proteolytic processing of APP yielding more amyloidogenic peptides. APP can be processed by at least 3 secretases: alpha-, beta-, and gamma-secretases. Beta-secretase initiates A-beta peptide generation by cleaving APP after Methionine 671 (APP770 numbering) leading to a 12 kd retained membrane carboxyterminal fragment (Citron M, Teplow D B, Selkoe D J. Generation of amyloid beta protein from its precursor is sequence specific. Neuron. 1995. 14:661–70). The 12 kd fragment may then undergo gamma-secretase cleavage within the hydrophobic transmembrane domain to release the 40, 42, or 43 residue A-beta peptides (Seubert P, Vigo-Pelfrey C, Esch F, Lee M, Dovey H, Davis D, Sinha S, Schlossmacher M, Whaley J, Swindlehurst C. Isolation and quantification of soluble Alzheimer's beta-peptide from biological fluids. Nature. 1992.359: 325–7).
Current treatments for AD provide only modest symptomatic relief. There is a real need for disease modifying agents that slow the course of the disease and prevent or delay the disease in susceptible individuals. The development of such agents requires, among others, progress in the understanding of the molecular basis of the disease and in the development of animal models.
The strategies used to reproduce the disease in animal models mainly reflects the divergent causes of AD: aging, APP and Psen FAD mutations.
A number of transgenic mouse lines overexpressing either human wildtype APP, or human APP with FAD mutations at the beta-secretase cleavage site (APP Swedish mutant; Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G. Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science. 1996. 274:99–102) and/or at the gamma-secretase cleavage site (APP London mutant; Games D, Adams D, Alessandrini R, Barbour R, Berthelette P, Blackwell C, Carr T, Clemens J, Donaldson T, Gillespie F. Alzheimer-type neuropathology in transgenic mice overexpressing V717F beta-amyloid precursor protein. Nature. 1995. 373:523–27) have been generated. These transgenic mouse lines have been crossed with transgenic lines overexpressing human Psen1 or Psen2 (gamma secretase activity) FAD mutants. Double transgenic mice show increased amyloid load but do not recapitulate all the aspects of the disease (for review see Van Leuven F. Single and multiple transgenic mice as models for Alzheimer's Disease. Progress in Neurobiology. 2000. 61, 305–312; Emilien G, Maloteaux J. M, Beyreuther K, and Masters C. L. Alzheimer Disease. Mouse models pave the way for therapeutic opportunities. Arch. Neurol. 2001. 57, 176).
Despite the fact that no FAD has up to now, been associated with mutation in human BACE (beta secretase activity) (Cruts M, Dermaut B, Rademakers R, Roks G, Van den Broeck M, Munteanu G, van Duij C M, Van Broeckhoven C. Amyloid beta secretase gene (BACE) is neither mutated in nor associated with early-onset Alzheimer's disease. Neurosci Lett. 2001. 313:105–7), BACE remains an attractive key player in the AD pathology. As a matter of fact, mice deficient in BACE (Cai H, Wang Y, Mc Carthy D, Wen H, Borchelt D. R, Price D. L, and Wong P. C. BACE1 is the major beta-secretase for generation of A-beta peptides by neurons. Nat. Neuroscience 2001. 4, 233; Luo Y, Bolon B, Kahn S, Bennett B D, Babu-Khan S, Denis P, Fan W, Kha H, Zhang J, Gong Y, Martin L, Louis J C, Yan Q, Richards W G, Citron M, Vassar R. Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci. 2001. 3:231–2; Roberds SL, Anderson J, Basi G, Bienkowski M J, Branstetter D G, Chen K S, Freedman S B, Frigon N L, Games D, Hu K, Johnson-Wood K, Kappenman K E, Kawabe T T, Kola I, Kuehn R, Lee M, Liu W, Motter R, Nichols N F, Power M, Robertson D W, Schenk D, Schoor M, Shopp G M, Shuck M E, Sinha S, Svensson K A, Tatsuno G, Tintrup H, Wijsman J, Wright S, McConlogue L. BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet. 2001. 10:1317–24) have been shown to lack A-beta peptides in the brain demonstrating the absolute need of BACE for the cleavage of APP. These findings also increase the validity of BACE as a drug target for AD.
Recently mice overexpressing human APP with FAD mutation at the beta-secretase site (APP Swedish) and overexpressing human BACE (beta secretase activity) have been reported (Chiocco M J and Lamb B T. Generation and characterization of genomic-based beta-secretase transgenic mice. Soc. Neurosci. Abstr. No. 13647. 2001. 27; Bodendorf U, Sturchler-Pierrat C, Christnacher A, Sommer B, Staufenbiel M, and Paganetti P. Mice transgenic for human BACE show increased beta-secretase activity in vivo. Soc. Neurosci. Abstr. No. 13445. 2001. 27).
The overexpression of BACE together with APP Swedish is redundant in the sense that both proteins target the same metabolic step of APP. As a matter of fact, it has been shown that BACE overexpression results in increased beta-secretase cleavage of both wild-type APP and Swedish APP but increased secretion of amyloid peptides is only observed with the wild-type, but not with the Swedish APP suggesting that gamma-secretase level is inadequate to process the amount of C99 fragment produced in case of APP Swedish mutant (Vassar R, Bennett B D, Babu-Khan S, Kahn S, Mendiaz E A, Denis P, Teplow D B, Ross S, Amarante P, LoeloffR, Luo Y, Fisher S, Fuller J, Edenson S, Lile J, Jarosinski M A, Biere A L, Curran E, Burgess T, Louis J C, Collins F, Treanor J, Rogers G, Citron M. Beta-secretase cleavage of Alzheimer's amyloid precursor protein by the transmembrane aspartic protease BACE. Science. Oct. 22, 1999;286(5440):735–41).